In eukaryotic cells, mitosis is initiated following the activation of a protein kinase known as “M-phase promoting factor” (MPF; also known as the H-phase specific histone kinase, or more simply as the H-phase kinase) This kinase consists of at least three subunits: the catalytic subunit (cdc2), a regulatory subunit (cyclin B) and a low molecular weight subunit (p13-Sucl) (Brizuela, L. et al., EMBO J. 6:3507–3514 (1987); Dunphy, W. et al., Cell 54:423–431 (1988); Gautier, J. et al., Cell 54:433–439 (1988); Arion, D. et al., Cell 55:371–378 (1988); Draetta, G. et al., Cell 56:829–838 (1989); Booher, R. et al., Cell 58:485–497 (1989); Labbe, J- C. et al. EMBO J. 8:3053–3058 (1989); Meijer, L. et al., EMBO J. 8:2275–2282 (1989); Gautier, J. et al., Cell 60:487–494 (1990); Gautier, J. and J. Maller, EMBO J. 10:177–182 (1991)). cdc2 and related kinases also associate with other cyclins (Giordana, A. et al., Cell 58:981–990 (1989); Draetta, G. et al., Cell 56:829–838 (1989); Richardson, H. E. et al., Cell 59:1127–1133 (1989)), and comprise a family of related enzymes that act at various stages of the division cycle (Paris, J. et al., Proc. Natl. Acad. Sci. USA 88:1039–1043 (1990); Elledge, S. J. and M. R. Spottswood, EMBO J. 10:2653–2659 (1991); Tsai, L- H. et al., Nature 353:174–177 (1991)).
The cdc2/cyclin B enzyme is subject to multiple levels of control. Among these, the regulation of the catalytic subunit by tyrosine phosphorylation is the best understood. In a variety of eukaryotic cell types, cdc2 is one of the most heavily tyrosine phosphorylated proteins (Draetta, G. et al., Nature 336:738–744 (1988); Dunphy, W. G. and J. W. Newport, Cell 58:181–431 (1989); Morla, A. O. et al., Cell 58:193–203 (1989)). Phosphorylation of the tyrosine 15 and also threonine 14 residues of cdc2 is regulated, in part, by the accumulation of cyclin above a threshold level at which association with cdc2 occurs (Solomon, M. J. et al., Cell 63:1013–1024 (1990)). Tyrosine phosphorylation inhibits the cdc2/cyclin B enzyme, and tyrosine dephosphorylation, which occurs at the onset of mitosis, directly activates the pre-MPF complex (Gautier J. et al., Nature 339:626–629 (1989); Labbe, J. C. et al., EMBO J. 8:3053–3058 (1989); Morla, A. O. et al., Cell 58:193–203 (1989); Dunphy, W. G. and J. W. Newport, Cell 58:181–431 (1989); Morla, A. O. et al., Cell 58:193–203 (1989); Gould, K. and P. Nurse, Nature 342:39–45 (1989); Jessus, C. et al., FEBS LETTERS 266:4–8 (1990)).
Given the role of cdc2 dephosphorylation in activation of MPF, there is much interest in the regulation of the cdc2 phosphatase. Genetic studies in fission yeast have established that the cdc25 gene function is essential for the initiation of mitosis (Nurse, P. et al., Mol. Gen. Genet. 146:167–178 (1976). The cdc25 gene product serves as a rate-determining activator of the cdc2 protein kinase (Russell, P. and P. Nurse, Cell 45:145–153 (1986); Ducommun, B. et al., Biochem. Biophys. Res. Common. 167:301–309 (1990); Moreno, S. et al., Nature 344:549–552 (1990)). Moreover, the mutant cdc2-F15, whose product cannot be phosphorylated on tyrosine, bypasses the requirement for cdc25 protein function (Gould, K. and P. Nurse, Nature 342:39–45 (1989)). Additional work has suggested that cdc25 is the cdc2 phosphatase. (Kumagai, A. and W. G. Dunphy, Cell 64:903–914 (1991); Strausfeld, U. et al., Nature 351:242–245 (1991)) and that cdc25 is the cdc2 phosphatase which dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation. (Dunphy, W. G. and A. Kumagai, Cell 67:189–196 (1991); Gautier, J. et al., Cell 67:197–211 (1991)).
The universal intracellular factor MPF triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. Little is known about the signals which control or determine timing of MPF activation and entry into mitosis or about ways in which those signals can be blocked or enhanced, resulting in inhibition or facilitation of entry into mitosis.
Because the signals that control dephosphorylation of cdc2 on tyrosine and threonine play a key role in controlling timing of MPF activation and entry into mitosis, there is great interest in the proteins which control cdc2 dephosphorylation. Further knowledge of these proteins and their regulatory functions would be useful because it would provide a basis for a better understanding of cell division and, possibly, an approach to altering how it occurs.